The bio-processing industry has traditionally used stainless steel systems and piping in manufacturing processes for fermentation and cell culture. These devices are designed to be steam sterilized and reused. Cleaning and sterilization are however costly labour-intensive operations. Moreover, the installed cost of these traditional systems with the requisite piping and utilities is often prohibitive. Furthermore, these systems are typically designed for a specific process, and cannot be easily reconfigured for new applications. These limitations have led to adoption of a new approach over the last ten years—that of using plastic, single-use disposable bags and tubing, to replace the usual stainless steel tanks.
In particular bioreactors, traditionally made of stainless steel, have been replaced in many applications by disposable bags which are rocked to provide the necessary aeration and mixing necessary for cell culture. These single-use bags are typically provided sterile and eliminate the costly and time-consuming steps of cleaning and sterilization. The bags are designed to maintain a sterile environment during operation thereby minimizing the risk of contamination.
Commonly used bags are of the “pillow style,” mainly because these can be manufactured at low cost by seaming together two flexible sheets of plastic. Three-dimensional bags have also been described, where further sheets may be used to create wall structures.
One of the successful disposable bioreactor systems uses a rocking table on to which a bioreactor bag is placed. The bioreactor bag is partially filled with liquid nutrient media and the desired cells. The table rocks the bag providing constant movement of the cells in the bag and also efficient gas exchange from the turbulent air-liquid surface. The bag, typically, has at least one gas supply tube for the introduction of air, carbon dioxide, nitrogen or oxygen, and at least one exhaust gas tube to allow for the removal of respired gases. Nutrients can be added through other tubes.
When cells are cultured at high densities, the aeration may not be sufficient to supply the cells. Bags with baffles along the edges to improve the mixing have been described in US 2010/0203624 and U.S. Pat. No. 7,195,394 but these are not sufficient to reach the cell densities desired today. Accordingly there is a need for improved aeration in rocking table bioreactors.
Traditionally, cell culture has been operated in a batch mode. In batch operation, the bioreactor is seeded with culture medium and a small amount of cells and the cells are grown to higher density without any further nutrient supply. The cells eventually die due to lack of nutrients or to build-up of toxic metabolites, and this will ultimately determine at which point harvest takes place. This method has several drawbacks—firstly, critical nutrients may become depleted leading to low final cell densities and consequently lower product yields; secondly, formation and quality of a potential recombinant protein product is often impaired due to the build-up of toxic metabolic by-products.
An alternative mode of operation is fed-batch. It involves culture initiation in a basal medium, and at a certain time point starting a supplementation, feed, of specific nutrients that otherwise may become limited. These nutrients can be added separately, or in groups, and they can be supplemented continuously or batch-wise. This strategy will counter-act early nutrient depletion and the implication is to prolong the culture time, increase the max cell density and thereby increase the potential for a high yield of e.g. a recombinant protein produced by the cells. However, a fed-batch strategy does not generally hinder the build-up of toxic metabolites, and this will result in conditions that are changing during the course of the culture. These changes may adversely affect the quality of the product, e.g. in terms of a less favourable glycosylation pattern.
It has long been recognized that perfusion culture offers better economics for certain processes. In this operation, cells are retained in the bioreactor, and toxic metabolic by-products are continuously removed. Feed, containing nutrients is continually added. This operation is capable of achieving high cell densities and more importantly, the cells can be maintained in a highly productive state for weeks—months. This achieves much higher yields and reduces the size of the bioreactor necessary. It is also a useful technique for cultivating primary or other slow growing cells. Perfusion operations have tremendous potential for growing the large number of cells needed for human cell and genetic therapy applications. A new perfusion process with a strategy to retain not only the cells in the bioreactor vessel, but also the product, has recently been described and this has gained a certain interest in the bioprocess community. In this process, a hollow fiber filter with a low cut-off pore size is used to separate high molecular particles such as cells and target proteins such as antibodies, from low molecular waste products. The high molecular particles remain in the retentate and thus in the reactor and the low molecular weight ones are continuously removed. This strategy results in not only high cell densities but also very high product concentrations in the reactor vessel.
Another recent development in perfusion cultivation is the alternating tangential flow (ATF) method described in U.S. Pat. No. 6,544,424.
Perfusion culture in inflatable bags has been described in US 2011/0020922 and U.S. Pat. No. 6,544,788, both of which discuss filters to be applied inside the bag for removal of filtrate through tubing. This arrangement puts severe constraints on the filter construction and there is a significant risk of filter clogging. Moreover, if the filter clogs then the whole culture has to be transferred to a new inflatable bag. Hence there is a need for improved constructions to be used in flexible bag perfusion culture.